IL-21 Promotes CD4 T Cell Responses by Phosphatidylinositol 3-Kinase–Dependent Upregulation of CD86 on B Cells

The Journal of Immunology

Kesley Attridge,*,1 Rupert Kenefeck,† Lukasz Wardzinski,*,† Omar S. Qureshi,* Chun Jing Wang,† Claire Manzotti,* Klaus Okkenhaug,‡ and Lucy S. K. Walker*,†

The cytokine IL-21 is a potent immune modulator with diverse mechanisms of action on multiple cell types. IL-21 is in clinical use to promote tumor rejection and is an emerging target for neutralization in the setting of autoimmunity. Despite its clinical potential, the biological actions of IL-21 are not yet fully understood and the full range of effects of this pleiotropic cytokine are still being uncovered. In this study, we identify a novel role for IL-21 as an inducer of the costimulatory ligand CD86 on B lymphocytes. CD86 provides critical signals through T cell–expressed CD28 that promote T cell activation in response to Ag engagement. Expression levels of CD86 are tightly regulated in vivo, being actively decreased by regulatory T cells and increased in response to pathogen- derived signals. In this study, we demonstrate that IL-21 can trigger potent and sustained CD86 upregulation through a STAT3 and PI3K-dependent mechanism. We show that elevated CD86 expression has functional consequences for the magnitude of CD4 T cell responses both in vitro and in vivo. These data pinpoint CD86 upregulation as an additional mechanism by which IL-21 can elicit immunomodulatory effects. The Journal of Immunology, 2014, 192: 2195–2201.

nterleukin-21 is known to influence multiple parameters of Alongside effects on B cells, several studies have also reported the immune response. The clinical importance of this path- that IL-21 promotes T cell activation. Pre-exposure to IL-21 has I way was first appreciated nearly a decade ago with the dem- been shown to increase the Ag responsiveness of CD8 T cells (14) onstration that IL-21 could augment antitumor immunity (1, 2), and and permit triggering by weak TCR agonists (15). CD4 T cell this has since become an active area of research (3–5). In addition to responses can also be augmented by IL-21, in part due to its abil- augmenting immunity against tumors, IL-21 signaling can directly ity to counteract regulatory T cell suppression (16, 17). The mech- induce apoptotic pathways in chronic lymphocytic leukemia (CLL) anisms by which IL-21 directly or indirectly promotes T cell B cells (6, 7) and diffuse large B cell lymphoma (8). responses are not yet fully defined. In this study we identify The role of IL-21 in T cell–dependent B cell responses has been a novel role for IL-21 in upregulating the expression of the co- extensively documented. IL-21 critically regulates Ab production, stimulatory ligand CD86 on B cells. We show that this requires partly in cooperation with IL-4 (9), and it promotes plasma cell activation of the PI3K pathway and is dependent on the PI3K differentiation in both mice (10) and humans (11). The intimate subunit p110d, a molecule currently being targeted in the setting interaction between follicular helper T (TFH) cells and germinal of several B cell malignancies (CLL, non-Hodgkin lymphoma) center B cells is also shaped by provision of IL-21; TFH cell– (18). The increased expression of CD86 on B cells is shown to derived IL-21 directly targets germinal center B cells, reinforcing have functional consequences for T cell expansion both in vitro their fate decision by sustaining bcl6 expression (12, 13). and in vivo. Collectively, these data suggest an additional mechanism by which IL-21 may augment adaptive immune responses and reveal a further level of T cell/B cell interaction directed by *Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham B15 2TT, United Kingdom; †Institute of Immunity and this cytokine. Transplantation, University College London Medical School, London NW3 2PF, United Kingdom; and ‡Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Cambridge CB22 3AT, United Kingdom Materials and Methods 1Current address: Kennedy Institute of Rheumatology, University of Oxford, Oxford, Mice U.K. DO11.10 TCR transgenic and BALB/c mice were purchased from The Received for publication August 6, 2013. Accepted for publication December 28, Jackson Laboratory. IL-21R2/2 mice were provided by Manfred Kopf 2013. (ETH Zurich) and were bred with DO11.10 TCR transgenic mice to 2 2 This work was supported by a Medical Research Council Non-Clinical Senior Fel- generate IL-21R / DO11.10 TCR transgenic progeny. p110dD910A mice lowship held by L.S.K.W. K.A. is funded by the Medical Research Council and C.M. were provided by K.O. Mice were housed at the University of Birmingham is funded by a Wellcome Trust project grant to L.S.K.W. R.K. and L.W. are funded by Biomedical Services Unit or at the University College London and used Diabetes U.K. according to Home Office and institutional guidelines. Address correspondence and reprint requests to Prof. Lucy S. K. Walker. Institute of Immunity and Transplantation, University College London Medical School, Royal Flow cytometry Free Campus, London NW3 2PF, U.K. E-mail address: [email protected] Cells were stained with mAbs against CD25 (PC61.5; eBioscience), CD4 The online version of this article contains supplemental material. (LT34; eBioscience), CD19 (1D3), CD86 (GL1; eBioscience), CD80 (16- Abbreviations used in this article: CLL, chronic lymphocytic leukemia; DC, dendritic 10A1), pSTAT1 (14/P-STAT1), pSTAT3 (49/P-STAT3), pSTAT5 (clone 47), cell; TFH cell, follicular helper T cell. and DO11.10 TCR (KJ1.26; eBioscience). All Abs were purchased from This is an open-access article distributed under the terms of the CC-BY 3.0 Unported BD Biosciences unless otherwise indicated. For pSTAT staining, cells were license. fixed in 4% paraformaldehyde for 10 min and permeabilized with 100% ice-cold methanol for 30 min. Statistics were performed using an unpaired Copyright Ó 2014 The Authors 0022-1767/14 two-tailed t test with a 95% confidence interval. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302082 2196 IL-21 TRIGGERS PI3K-DEPENDENT CD86 UPREGULATION

Short-term splenocyte cultures BALB/c splenocytes (1 3 105) were cultured for 15–16 h alone, with IL-21 at 25, 50, 100 or 200 ng/ml (PeproTech), or with 1 mg/ml LPS (Sigma- Aldrich). For time course experiments, cells were harvested at 2, 4, 6, 8, or 15 h. Short-term B cell cultures Magnetic separation (Miltenyi Biotec) was used to purify CD19+ B cells from BALB/c or p110dD910A spleen. Cells (1 3 106) were cultured for 16 h alone or in the presence of 200 ng/ml IL-21 (PeproTech) or 10 ng/ml IL-4 (PeproTech). For STAT3 inhibition experiments cultures were supplemented with 10, 50, or 100 mM S3I-201 (Calbiochem) as indicated. For PI3K inhibition experiments cultures were supplemented with 10 mMLY- 294002 (Invitrogen) as indicated. For assessment of activated STAT proteins cells were cultured for 2 h alone or in the presence of 200 ng/ml IL- 21 (PeproTech). For experiments to determine the target of IL-21, 2.5 3 104 BALB/c or IL-21R2/2 B cells were cultured with 2.5 3 104 mag- netically separated (Miltenyi Biotec) CD4+CD252 T cells from BALB/c or IL-21R2/2 lymph node for 16 h alone or in the presence of 200 ng/ml IL- 21 (PeproTech). Confocal microscopy Magnetic separation (Miltenyi Biotec) was used to purify CD19+ B cells from BALB/c spleen. Cells (1 3 106) were cultured for 6 h alone, with 200 ng/ml IL-21 (Peprotech), with 10 mg/ml cycloheximide (Sigma-Aldrich), or with both. Cells were stained with mAb against CD86 (GL1; eBioscience) and imaged using glass bottom culture dishes (MatTek). Imaging was carried out using a 3100 oil immersion objective. RT-PCR Magnetic separation (Miltenyi Biotec) was used to purify CD19+ B cells from BALB/c spleen. mRNA was isolated at this point or after culture of 1 3 106 cells for 16 h alone or in the presence of 200 ng/ml IL-21 (PeproTech). Quantitative PCR was performed to assess the expression of b2-microglobulin (Eurofins MWG Operon) or CD86 (TaqMan gene expression assay; Applied Biosystems). In vitro proliferation assays Magnetic separation (Miltenyi Biotec) was used to purify CD4+CD252 T cells from IL-21R2/2 lymph node. Cells (2.5 3 104) were cultured with 2.5 3 104 CD19+ B cells from BALB/c or IL-21R2/2 spleen, with 0.8 mg/ml anti-CD3 (BD Biosciences) alone or in the presence of 10 mg/ml anti-CD86 (Bio X Cell). Triplicate wells were pooled and harvested at day 1 to assess CD86 expression or day 3 to determine cell counts by flow cytometry. Adoptive transfers CD19+ B cells from BALB/c or IL-21R2/2 spleen were isolated by magnetic separation (Miltenyi Biotec) and cultured for 16 h with 1 mg/ml OVA peptide. Peptide-pulsed cells (3 3 106) were injected i.v. into IL- 21R2/2 recipients. Magnetic separation (Miltenyi Biotec) was used to purify CD4+ cells from IL-21R2/2 DO11.10 TCR+ lymph node. Twenty- four hours after B cell transfer, a total of 2 3 106 CellTrace Violet–labeled IL-21R2/2 DO11.10 TCR+ T cells were injected i.v. and 1 mgIL-21 (PeproTech) or PBS i.p. Where indicated recipients were also given 100 mg anti-CD86 (Bio X Cell) or PBS i.p. immediately after T cell transfer. Mice were culled at day 7 for analysis of inguinal lymph node and splenic cells. FIGURE 1. IL-21 upregulates CD86 expression on B cells. (A)BALB/c Results splenocytes (1 3 105) were cultured for 16 h alone or in the presence of 200 IL-21 upregulates CD86 expression on B cells ng/ml IL-21. Plots show representative CD80 and CD86 expression for gated CD19+ Bcells.(B) BALB/c splenocytes (1 3 105) were cultured for 16 h To explore the influence of IL-21 on costimulatory ligand ex- alone or in the presence of the indicated doses of IL-21. Histograms show pression, murine splenocytes were cultured in the presence or CD86 expression for gated CD19+ B cells and graph shows collated CD86 absence of IL-21 for 16 h and the expression of CD86 and CD80 mean fluorescence intensity (MFI) data. (C) BALB/c splenocytes (1 3 105) was assessed by flow cytometry. We noted a marked elevation of were cultured alone or in the presence of 200 ng/ml IL-21 and harvested at the + CD86, but not CD80, on CD19+ B cells in the timeframe examined indicated time points. Graph shows collated CD86 MFI data for gated CD19 D 3 5 (Fig. 1A). Titration (Fig. 1B) and time course (Fig. 1C) data Bcells.( ) BALB/c splenocytes (1 10 ) were cultured for 16 h alone or in established that IL-21–mediated CD86 upregulation was dose- the presence of either 200 ng/ml IL-21 or 1 mg/ml LPS. Graph shows collated CD86 MFI data for gated CD19+ B cells. Data are representative of at least dependent and appeared maximal at ∼8 h following stimulation. three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. The magnitude of CD86 upregulation observed in response to IL- 21 was comparable, if not greater, than that seen with a 1 mg/ml dose of LPS, a TLR ligand known to drive CD86 upregulation CD86 expression at baseline and this was not further elevated by (Fig. 1D). In contrast, splenic dendritic cells (DCs) showed some IL-21 under the conditions assessed (Supplemental Fig. 1). The Journal of Immunology 2197

CD86 upregulation requires direct signaling to B cells and CD86 upregulation requires STAT3 and PI3K protein neosynthesis IL-21 signaling is known to involve STAT3 activation in both Because the above assays did not use purified B cells, it re- mouse (22) and human (23, 24) B cells, and the ability of STAT3 mained possible that IL-21 was altering CD86 expression in- to mediate IL-21 effects has been demonstrated using B cells directly by acting on a different cell type. In this regard we have isolated from STAT3-deficient patients (25). We confirmed the previously shown that conventional CD4 T cells express high ability of IL-21 to activate the STAT3 pathway in B cells (Fig. 3A) levels of IL-21R and can respondtoIL-21byacquiringresis- and then sought to determine whether this pathway was involved tance to regulatory T cell suppression (19). To dissect whether in IL-21–mediated CD86 upregulation. In the presence of the STAT3 regulation of CD86 expression involved direct signaling to B cells inhibitor S3I-201, IL-21 was no longer able to induce significant or was an indirect consequence of IL-21 signaling to T cells, CD86 upregulation (Fig. 3B, 3C), suggesting a critical role for we took advantage of mice genetically deficient for the IL-21R STAT3inthisprocess. (20). Cocultures of T cells and B cells were established in which A second major pathway triggered by IL-21 is the activation of the cells derived from either wild-type or IL-21R–deficient mice the enzyme PI3K (22, 26), and the ability of BCR engagement to and these were incubated for 16 h with or without IL-21. As ex- upregulate CD86 is known to be dependent on PI3K signaling (27). pected, when both T and B cells expressed IL-21R, IL-21 up- To determine whether IL-21 upregulates CD86 via a PI3K-dependent regulated CD86 expression (Fig. 2A, far left panels)andwhen pathway, we first employed the PI3K inhibitor LY-294002. Strikingly, neither cell type expressed IL-21R, CD86 was unchanged (Fig. IL-21–mediated CD86 upregulation was completely abrogated in the 2A, second panels). Crucially, in situations where B cells were presence of this inhibitor (Fig. 4A). The related cytokine IL-4 IL-21R+/+ but T cells were IL-21R2/2, addition of IL-21 retained retained the ability to upregulate CD86 in the presence of the PI3K the capacity to upregulate CD86 (Fig. 2A, third panels). Fur- inhibitor, consistent with previous studies (27). We next took ad- thermore, preventing B cells alone from receiving IL-21 signals vantage of mice expressing a catalytically inactive form of the PI3K completely abrogated the ability of IL-21 to upregulate CD86 p110d subunit (D910A mice) (28), because this subunit is known to (Fig. 2A, far right panels). Collectively, these data indicate that be important in the B cell lineage (29, 30). Purified B cells from direct signaling of IL-21 to B cells is responsible for mediating D910A animals failed to upregulate CD86 in response to IL-21, CD86 upregulation. whereas their ability to upregulate CD86 in response to IL-4 The upregulation of CD86 by the cytokine IFN-g has been remained intact (Fig. 4B). Collectively, these data indicate that reported to be independent of protein neosynthesis (21). To ex- IL-21 uses STAT3 and PI3K to drive CD86 upregulation and amine whether upregulation of CD86byIL-21requiredprotein pinpoint p110d as the critical PI3K subunit responsible. neosynthesis, we performed experiments using purified B cells in the presence or absence of cycloheximide. The ability of IL-21 to IL-21–mediated CD86 upregulation has functional upregulate B cell CD86 expression was completely abrogated in consequences in vitro the presence of cycloheximide, indicating a requirement for pro- To test whether the upregulation of CD86 in response to IL-21 had tein neosynthesis (Fig. 2B, 2C). We next assessed whether up- functional consequences, in vitro T cell proliferation assays were regulation of CD86 by IL-21 was transcriptionally regulated; this performed. CD4+CD252 T cells from IL-21R2/2 mice were used revealed that mRNA for CD86 was strongly upregulated by ex- to preclude direct effects of IL-21 on the T cells themselves. IL- posure to IL-21 (Fig. 2D). Taken together, these data indicate that 21R2/2 T cells were activated by anti-CD3 in the presence of de novo transcription and translation of CD86 mRNA is required B cells that were either wild-type or IL-21R deficient; in this for IL-21–mediated CD86 upregulation. setting any IL-21 made by the T cells would potentially be able to

FIGURE 2. Induction of CD86 by IL-21 requires direct signaling to B cells and is dependent on protein neosynthesis. (A) BALB/c CD4+CD252 conventional T cells (2.5 3 104) were cultured with 2.5 3 104 CD19+ B cells for 16 h alone or in the presence of 200 ng/ml IL-21. Cell populations were deficient for the IL- 21R as indicated. Plots show CD86 expression for gated CD19+ B cells. (B) BALB/c CD19+ B cells (1 3 106) were cultured for 6 h alone, with 200 ng/ml IL-21, with 10 mg/ml cycloheximide, or both. Representative confocal microscopy images show CD86 staining. Scale bars, 10 mm. (C)GraphshowscollatedCD86meanfluo- rescence intensity (MFI) for cells from (B) analyzed by flow cytometry. Data are representative of three independent experiments. (D) BALB/c CD19+ B cells (1 3 106) were cultured for 16 h alone or in the presence of 200 ng/ml IL-21. Graph shows relative CD86 mRNA expression for cultured cells and freshly isolated CD19+ B cells. Data are representative of three independent experiments. **p , 0.01. 2198 IL-21 TRIGGERS PI3K-DEPENDENT CD86 UPREGULATION

FIGURE 4. Induction of CD86 by IL-21 is dependent on PI3K p110d. A + 3 6 FIGURE 3. Induction of CD86 by IL-21 is dependent on STAT3 activa- ( ) BALB/c CD19 B cells (1 10 ) were cultured for 16 h alone or in the tion. (A) BALB/c CD19+ B cells (1 3 106) were cultured for 2 h alone or in presence of 200 ng/ml IL-21 or 10 ng/ml IL-4. Where indicated, cultures the presence of 200 ng/ml IL-21. Histograms show representative staining for also contained 10 mM LY-294002. Histograms show CD86 expression for + B + 3 6 phosphorylated STAT proteins as indicated. (B) BALB/c CD19+ Bcells(13 gated CD19 B cells. ( ) BALB/c CD19 B cells (1 10 ) were cultured 106) were cultured for 16 h alone or in the presence of 200 ng/ml IL-21, for 16 h alone or in the presence of 200 ng/ml IL-21 or 10 ng/ml IL-4. 100 mM S3I-201, or both. Histograms show representative CD86 expression Cells expressed the wild-type or mutant isoforms of PI3K P110d as indi- + for gated CD19+ Bcells.(C) Graph shows collated CD86 expression data for cated. Histograms show CD86 expression for gated CD19 B cells. Data cells from B when cultured with the indicated doses of S3I-201. Data are are representative of three independent experiments. representative of three independent experiments. *p , 0.05, **p , 0.01. stimulate the proliferation of Ag-specific CD4 T cells. Wild-type 2/2 2/2 upregulate CD86 on the wild-type B cells but not the IL-21R or IL-21R B cells were pulsed with OVA323–339 peptide and ones. In line with this, CD86 was upregulated to a greater extent adoptively transferred into IL-21R2/2 hosts. OVA-specific T cells on the IL-21R+/+ B cells than on the IL-21R2/2 ones (Fig. 5A). IL-4 (DO11) that were also IL-21R2/2 were CellTrace labeled and did not appear to contribute to CD86 upregulation in this setting as injected into the same hosts, so that their response to the OVA- assessed by inclusion of blocking anti–IL-4 Ab (data not shown). pulsed B cells could be tracked. To maximize the effects of IL-21 The increased CD86 expression on IL-21R+/+ B cells was associated on B cell phenotype, rIL-21 was injected i.p. where indicated. with significantly greater T cell proliferation (Fig. 5B) and activa- Ensuring that the host animals and the adoptively transferred tion marker expression (Fig. 5C and data not shown). T cell pro- T cells were IL-21R2/2 allowed us to restrict the IL-21 effects to liferation and activation marker expression was dependent on CD86 the B cell compartment. At day 7, single-cell suspensions from as demonstrated by Ab blockade of this pathway (Fig. 5B, 5C). inguinal lymph node and spleen were stained by flow cytometry to identify the adoptively transferred Ag-specific T cells and assess IL-21–mediated CD86 upregulation has functional their proliferation status (Fig. 6A). CellTrace profiles for gated consequences in vivo Ag-specific T cells revealed greater proliferation in mice that had To extend the above observations to an in vivo setting, we es- received IL-21R+/+ B cells compared with those that had received tablished a system in which peptide-pulsed B cells were used to IL-21R2/2 B cells. Proliferation was particularly evident in the The Journal of Immunology 2199

numbers of Ag-speciﬁc T cells (Fig. 6B). The ability of IL-21 signaling to B cells to augment the T cell response was dependent on CD86 as assessed by in vivo Ab blockade (Fig. 6C). Recovery of injected B cells at day 1 conﬁrmed increased expression of CD86 on IL-21R+/+ B cells compared with IL-21R2/2 B cells (Supplemental Fig. 2). Collectively, these data demonstrate that IL-21 increases expression of CD86 on B cells and that this increased availability of costimulatory ligand has functional consequences for the magnitude of T cell responses in vitro and in vivo.

FIGURE 5. IL-21 signaling to B cells promotes CD86-dependent T cell Discussion proliferation. IL-21R2/2 CD4+CD252 conventional T cells (2.5 3 104) 2 2 IL-21 is recognized to exert multiple effects on the differentiation were cultured with 0.8 mg/ml anti-CD3 and 2.5 3 104 IL-21R / or IL- and function of B cells. Although first recognized for its ability to 21R+/+ CD19+ B cells alone or in the presence of 10 mg/ml anti-CD86. (A) Graph shows collated CD86 mean fluorescence intensity (MFI) data for promote human B cell proliferation (31), it can also trigger B cell gated CD19+ B cells harvested after 16 h in the absence of anti-CD86 apoptosis (32) with the outcome of IL-21R signaling most likely blocking Ab. (B) Graph shows collated data for absolute CD4+ conven- dictated by its context (10, 33). IL-21 has a well-established role tional T cell counts after harvest at day 3 when cultured with IL-21R2/2 or in the induction of plasma cell differentiation (10, 11) and this IL-21R+/+ B cells as indicated. (C) Graph shows collated CD25 MFI data involves the cooperative binding of STAT3 and IFN regulatory for CD4+ conventional T cells from (B). *p , 0.05, **p , 0.01. factor 4 to the IL-21 response element in the Prdm1 gene encoding Blimp-1 (34). Intriguingly, IL-21 also promotes the expression of Bcl6 spleen, consistent with the likely trafficking of the adoptively a key transcriptional regulator of germinal center B cell differ- transferred peptide-pulsed B cells to this site. The enhanced T cell entiation (12, 13), despite the fact that Blimp-1 and Bcl6 have +/+ response to IL-21R B cells was also reflected in higher absolute mutually antagonistic effects. Thus IL-21 acts at multiple stages to modulate B cell activation, differentiation, and death. Our recent findings provide additional insight into the effects of IL-21 on B cells, demonstrating an upregulation of CD86 that has consequences for T cell–dependent immune responses. The role of IL-21 in DCs is less well studied and our side-by-side comparison of B cells and DCs suggested CD86 was not obviously upregulated by IL-21 in the latter population. Whereas it has been shown that IL-21 can inhibit DC maturation (35, 36), other studies suggested IL-21 can enhance DC function (37), and recent analysis has identified a role for IL-21 in triggering DC migration in a virus-induced diabetes model (38). Interestingly, in the latter study, reduced migration of IL-21R2/2 DCs was accompanied by decreased CD86 expression. Thus, further work is required to fully elucidate the biological function of IL-21 in DCs. The provision of CD28 costimulation is known to be of critical importance in thresholding T cell responses. As a consequence, stimuli that alter expression of the costimulatory ligands CD86 and CD80 are likely to have a significant impact on T cell immunity. This is particularly true of CD86, as it is thought to be the dominant ligand for driving T cell responses (39). Expression of CD86 and CD80 is tightly regulated, and the CD28 homolog CTLA-4 serves to limit their availability by competitive inhibition and ligand downregulation (40–42). Unregulated availability of ligands, in mice lacking CTLA-4, results in unfettered T cell responses that culminate in lethal autoimmunity (43, 44). Thus, restricting CD86 and CD80 expression represents a major control point for adaptive immunity. Counteracting the downregulation of ligand by CTLA-4, various inflammatory stimuli serve to enhance ligand expression. FIGURE 6. IL-21 signaling to B cells promotes CD86-dependent T cell Accordingly, TLR ligands and cytokines such as IFN-g have been proliferation in vivo. (A) OVA peptide-loaded IL-21R2/2 or IL-21R+/+ shown to upregulate CD86 expression. In this study, we identify IL- 2 2 CD19+ Bcells(33 106) were adoptively transferred into IL-21R / 21 as a potent upregulator of CD86 expression on B cells. 6 2/2 recipients. After 24 h, mice received 2 3 10 CellTrace-labeled IL-21R IL-21 is one of several cytokines associated with systemic au- DO11+ CD4+ Tcellsi.v.and1mg IL-21 i.p. Histograms show representative + + toimmunity (45) and has been linked with lupus pathogenesis in CellTrace dilution for gated CD4 DO11 T cells harvested from spleens or mice (10, 46) and humans (47). The capacity of IL-21 to upreg- inguinal lymph nodes of IL-21R2/2 or IL-21R+/+ B cell recipients at day 7. ulate CD86 and promote T cell costimulation could conceivably (B) Graph shows representative absolute CD4+DO11+ T cell counts from (A). (C)IL-21R2/2 T cells were activated in the presence of OVA peptide loaded contribute to its pathogenic effects. Consistent with this, B cell IL-21R2/2 or IL-21R+/+ CD19+ B cells (as above) and recipient mice re- expression of the costimulatory ligands CD86 and CD80 has been ceived 100 mg anti-CD86 or isotype control Ab i.p. on day 1. Graphs show shown to be essential for autoreactive T cell activation and de- collated absolute CD4+DO11+ T cell counts. Data are representative of at velopment of joint pathology in the proteoglycan-induced arthritis least two independent experiments. *p , 0.05. model in mice (48). 2200 IL-21 TRIGGERS PI3K-DEPENDENT CD86 UPREGULATION

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